In the current study, we used microglia/neuron co-cultures to document several important findings about the mechanisms by which activated microglia can produce neurodegenerative responses. First, the importance of microglia p38α MAPK signaling was demonstrated by the observations that neurons in co-culture with p38α-deficient microglia were protected against LPS-induced neurotoxicity, synaptic protein loss, and neurite degeneration. Second, p38α-dependent microglia TNFα production was shown to be involved in the mechanism of the LPS-induced neuron damage by the findings that p38α KO microglia produce much less TNFα in response to LPS compared to WT microglia, that adding back TNFα to p38α KO microglia increases the LPS-induced neurotoxicity, and that neutralization of TNFα in WT microglia decreases the LPS-induced neuron damage. Altogether, our results demonstrate the critical importance of the p38α MAPK signaling pathway and overproduction of the proinflammatory cytokine TNFα in the dysregulated microglia inflammatory responses to an LPS stressor, leading to microglia-induced neuronal dysfunction.
Our demonstration that microglia p38α MAPK signaling is important in the mechanism of LPS-induced neuron damage is consistent with numerous findings that have implicated p38 MAPK activation in the process of neuronal death in a variety of neurodegenerative disorders. In addition, our studies here using cell-selective, isoform-specific KO mice extend previous findings by showing that the p38α MAPK isoform in microglia is a key mediator of LPS-induced neuronal and synaptic dysfunction. We also provide evidence that one mechanism by which LPS activation of microglia p38α MAPK signaling leads to neuron death is through up-regulation of the proinflammatory cytokine TNFα.
The p38 MAPK family consists of four major isoforms (p38α, β, δ, γ) that have different cell and tissue expression patterns, substrate specificities, and functions [for reviews, see: [14, 28]]. The patterns of expression and activation of the p38α isoform in peripheral immune cells [29, 30] suggested that this isoform might play a major role in the inflammatory response. Early attempts using genetic KO approaches to explore the role of p38α in inflammatory responses were hampered because of embryonic lethality seen with global KO of p38α. However, a number of more recent studies have used conditional ablation of p38α in specific cell types to provide direct evidence that the p38α isoform is of central importance for many peripheral inflammatory responses, such as inflammation-induced arthritic bone loss , inflammatory skin injuries , inflammatory responses of myeloid cells in an experimental colitis model , immune cell recruitment and pathogen clearance in intestinal epithelial cells , and LPS-induced cytokine production in macrophages . These and other studies using selective p38α inhibitors and drug-resistant forms of the kinase have demonstrated the importance of p38α signaling in mediating peripheral inflammatory responses [34–37].
Although there is broad agreement that p38α plays a key role in cytokine production and other inflammatory responses in peripheral immune cells, the contribution of p38α to pathological microglial activation and detrimental inflammation in CNS disorders is less well understood. Increasing evidence suggests that p38 signaling cascades contribute to CNS cytokine overproduction and neurodegenerative sequelae [for reviews, see: [14, 38, 39]], but few studies have tested the specific role of microglia p38α. Expression of the p38α isoform in microglia was reported to increase early after transient global ischemia , and administration of p38 inhibitors reduced infarct volume [15, 41] and suppressed proinflammatory cytokine production . We recently demonstrated  a direct linkage between microglia p38α and proinflammatory cytokine production in response to different stressors by showing that inhibition of p38α in microglia with either a pharmacologic or genetic approach suppresses proinflammatory cytokine up-regulation induced by toll-like receptor ligands or beta-amyloid.
In the present study, we explored the consequences of the microglial p38α-dependent proinflammatory cytokine response on neuronal endpoints. By using microglia deficient in p38α, we showed definitively that microglial p38α is critical for LPS-induced neuron dysfunction and we implicated p38α-dependent production of the proinflammatory cytokine TNFα in the mechanism of neuron damage. The potential involvement of TNFα was not unexpected, as this proinflammatory cytokine has been shown to induce neurotoxicity in models of CNS neurodegenerative disorders [42–44], and blocking TNFα signaling can be neuroprotective [45, 46]. However, TNFα is pleiotropic and can also have neuroprotective functions [for review, see: ]. Multiple factors influence whether TNFα will exert neurotoxic or neuroprotective actions, including the level and duration of expression in a particular cell type or brain region, the microglia activation state, the particular disease or disease stage, the levels of different TNF receptors and adapter proteins, and the upstream activators and downstream effectors in the signaling pathways. Thus, it was somewhat surprising that microglia p38α-dependent production of TNFα in response to an LPS insult appeared to be sufficient to induce neuron death, as evidenced by the observations that anti-TNFα antibody treatment resulted in increased neuronal survival back to control values, and addition of TNFα to KO microglia reduced neuronal survival to the same levels as WT. Altogether, our data demonstrate that microglia p38α activation in response to an LPS stressor stimulus and the consequent dysregulated TNFα signaling can lead to neuron damage.
Of note is our finding that p38α MAPK deficiency in microglia attenuates LPS-induced loss of specific synaptic proteins in the co-cultures. Previous studies have shown a correlation between p38 MAPK activation and a decline in synaptophysin levels in AD transgenic mouse models and in primary microglia and cortical neuron co-cultures stimulated with LPS [48, 49], and pharmacological inhibition of p38α MAPK significantly reduced TNFα and IL-1β production and prevented synaptophysin loss in an AD mouse model . Our results here demonstrate for the first time a linkage of p38α MAPK and TNFα to LPS-induced decreases in SNAP25 and drebrin. Because drebrin, a postsynaptic protein found within dendritic spines, is important for spine morphogenesis and maintenance [50, 51], future studies should examine in more detail the mechanisms by which p38α MAPK influences dendritic pathology and synaptic deterioration such as seen in many neurodegenerative disorders. Future studies should also explore whether microglia p38α MAPK is involved in beneficial responses of activated microglia, as the current study focused only on detrimental consequences of microglia p38α activation.